In order to better understand the role of DNA replication fidelity in mutagenesis, we have isolated strains of E. coli that replicate their DNA with increased fidelity (antimutator mutants). They were obtained as suppressors of the high mutability of strains defective in postreplicative mismatch repair (mutL) or in exonucleolytic proofreading (mutD). In virtually all cases, the responsible mutation was found to reside in the dnaE gene, encoding the alpha subunit of DNA polymerase III responsible for replicating the bacterial chromosome. In addition to lowering the mutation rate in mutL strains by 3- to 20-fold, they were capable of lowering the mutation rate in an otherwise wild-type background by about 2-fold. This suggests that in E. coli uncorrected DNA replication errors may represent about one half of all spontaneous mutations. DNA sequencing of lacI forward mutations revealed that this one half is almost entirely composed of transversion base-pair substitutions, suggesting that these represent uncorrected replication errors. In contrast, the transitions may result from unrepaired DNA damages. We are also investigating the mechanisms by which the dnaE antimutators exert their effect. Our data suggest that the mutant DNA polymerases are either impaired in the forward going polymerization rate, allowing increased proofreading of polymerase errors, or are otherwise destabilized, prompting increased dissociation from terminal mismatches. We are also investigating whether dnaE antimutator effects occur equally in both DNA strands or are specific to either leading or lagging strands. Other antimutator mechanisms that are being investigated involve the loss of error-prone components, such as the SOS-associated DNA polymerases IV or V.